Dengue remains an international public health problem affecting urban populations in tropical and sub-tropical regions, where it is currently estimated that about 2.5 billion people are at risk of dengue infection. Dengue virus is a single positive-stranded RNA virus of the family Flaviviridae; genus Flavivirus, which is transmitted among humans primarily by Aedes aegypti mosquitoes. In humans, dengue infection can produce diseases of a wide spectrum of severity, from asymptomatic to flu-like dengue fever (DF), to life-threatening dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS). DHF is particularly associated with capillary leakage, hemorrhage, circulatory shock, and representing life-threatening complication.
Due to a number of factors, including increasing urbanization and globalization of travel, dengue disease is re-emerging in the Americas, where it has caused an estimated 890,000 cases, of which 26,000 were DHF (45). The mortality of DHF is age-dependent, primarily occurring in the children and the elderly (3, 45). In Southeast Asia, a disproportionate amount of DHF hospitalizations are of children whereas in the Americas, there is a more even distribution across ages.
The risk factors and etiology of DHF are not fully understood. There are four serotypes of dengue virus, and often a region may have more than one circulating serotype at a time. Many epidemiological studies have found an 40 to 80-fold increased risk of DHF after a second infection with a different serotype (3, 5, 6). This observation has led to the “antibody dependent enhancement theory,” which hypothesizes that neutralizing antibodies generated during the adaptive immune response cross-react, but do not neutralize, a second infecting dengue virus serotype. These antibody-viral complexes are taken up by immunocytes by binding the cell-surface Fc receptors. As a result, highly activated immunocytes release enhanced cytokines and factors involved in vascular leakage. Other evidence points to DHF being the result of an interplay between host and viral factors, including cell-mediated immunity (1).
Currently, there is no drug therapy or vaccine for DHF. However, early therapy aiming to treat individual symptoms can reduce mortality. Typical dengue treatments include transfusion of fresh blood or platelets to correct blooding, giving intravenous (IV) fluids and electrolytes to correct electrolyte imbalances and dehydration, and oxygen therapy to treat low blood oxygen. Although DHF fatality rates can exceed 20%, early identification and intensive supportive therapy can reduce the rate to less than 1% (7). Therefore, detection and differentiation of dengue disease severity early in the course of infection is critical for the prognosis and treatment of patients.
Currently diagnosis of dengue virus infection is made by physical examination of the patient and routine clinical laboratory tests such as complete blood count (CBC). A positive tourniquet test has been considered to be a sensitive parameter for dengue diagnosis. More than 90% of cases can be correctly diagnosed for dengue infection by taking into account of the patient's medical history, physical signs, and a positive tourniquet test. However, a definitive diagnosis for dengue infection requires laboratory confirmation, especially in regions where other endemic infectious diseases mimic the syndromes caused by dengue infection. Definitive diagnostic tests for dengue infection include isolation of viable virus, and identification of viral RNA in serum or plasma. Several factors limit routine application of these tests, including the timing of specimen collection, and the availability of equipment.
Serological techniques are also used in dengue diagnosis. Serological tests are commonly used in the field because timing of specimen collection is flexible, and immunoglobulins are not easily degraded or inactivated by harsh treatment of specimens. The most commonly used serological techniques for the diagnosis of dengue infection are the hemagglutination inhibition (HI) test, and immunoglobulin M or G (IgM or IgG) captured enzyme-linked immunosorbent assay (ELISA). Results from both IgM and IgG captured ELISA can be used to differentiate between the cases of primary and secondary dengue infection. In primary infection, the ratio of anti-dengue IgM to anti-dengue IgG is relatively high for at least a month following infection, but in secondary infection, a rapid increase of IgG antibody generally occurs following infection, and the ratio of anti-dengue IgM to anti-dengue IgG in a single acute specimen is low. U.S. Pat. No. 6,870,032 describes a method for early detection of a flavivirus-induced infection including dengue infection by detecting NS1 protein via enzyme linked immunosorbant assay (ELISA) technique employing at least two antibodies, i.e., a first capture antibody to capture the NS1, and a second antibody for detecting the presence of NS1 in biological samples. However, both HI test and IgG-captured/IgM-captured ELISA usually require paired acute and convalescent phase serum samples collected a week or more apart and a definitive diagnosis is made based on a fourfold rise in anti-dengue antibody. In general, current available dengue diagnostic assays do not allow detection and targeted treatment of DHF during early clinical period. A more rapid test with less reliability on equipment is needed.
Recent advances in global scale proteomics technologies enable the detection of candidate protein biomarkers. These biomarkers include proteins, peptides, or metabolites whose measurement alone (or in a combination) can be used to reliably indicate a disease outcome. With the advancement of multidimensional profiling techniques, the systematic and quick identification of predictive proteins associated with a disease have become feasible.
U.S. Pat. No. 7,939,287 to Tsimikas et al. describe a method of identifying a subject having or at risk of developing coronary artery disease using biomarkers. U.S. Pat. No. 7,608,406 to Valkirs et al. disclose a panel of biomarkers used in a method for early diagnosis and differentiation of stroke types and transient ischemic attacks and for determining prognosis of a patient presenting with stroke symptoms. U.S. Pat. No. 7,598,09 to Ray et al. teach methods for diagnosis of Alzheimer's disease by detecting a collection of proteinaceous biomarkers in blood samples.
U.S. Pat. No. 7,629,117 to Avirutnan, et al. disclose methods of determining risk of developing Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) in an individual infected with dengue virus (DV). The methods comprise determining, in a fluid or tissue sample of an individual, the presence, absence or quantity of dengue virus protein NS1, and determining, in a fluid or tissue sample of the individual, presence, absence or quantity of SC5b-9 complement complex. The methods can further comprise comparing the levels of NS1 protein and SC5b-9 complement complex with a database comprising epidemiological data correlating levels of NS1 protein and SC5b-9 complement complex with probability of developing DHF/DSS in a population. Complement activation is known to be a key pathogenic mechanism in dengue virus infection. Accelerated complement consumption and marked reduction of plasma complement components are observed in DSS patients during shock. However, the cause of complement activation has remained unknown. Terminal complement complex (SC5b-9) is a group of proteins in the terminal pathway of complement system. It is not always generated when the complement system is triggered due to a tightly-controlled set of the complement regulatory proteins. Only strong or efficient complement activators can successfully cause SC5-9 liberation. In healthy individuals, very low or insignificant level of terminal complement complex can be detected.
Despite the attempts of dengue diagnosis via the detection of selected biomarkers. Identification of predictive biomarkers in complex biofluids, such as plasma, has been challenging for proteomics technologies. Plasma is a complex biofluid, with its constituent proteins present in a broad dynamic concentration range spanning 6 log orders of magnitude or more (25, 26). Moreover, the tendency of high-abundance proteins to adsorb lower-abundance proteins and peptides (27, 28), the presence of proteases that may produce peptide fragments (29, 30), and the individual variation in plasma protein abundances serve to compound the difficulties in comprehensive proteomic analyses of plasma.